Live vaccine for the prevention of salmonellosis in water fowl, a process for making and applying the same

ABSTRACT

A novel live vaccine for the prevention of salmonellosis in water fowl comprising essentially a suspension of living culture of the attenuated strain Salmonella typhi-murium No. 3, deposited at the All-Union State Research Control Institute for Veterinary Preparations, the USSR Ministry of Agriculture (No. 121), in a concentration of 2-4 billion microbe cells per 10 cm 3  of drinking water. A process for making said vaccine, wherein the attenuated strain Salmonella typhi-murium No. 3 is cultivated in a culture medium containing sources of carbon, nitrogen, mineral salts, biologically active substances at a temperature of 37°-38° C. to produce the highest attainable accumulation of biomass during a period of 10-14 hours with subsequent drying and obtainment of the vaccine. 
     A method of specific prophylaxis of salmonellosis in water fowl, wherein the live vaccine is administered to the poultry twice orally at a 2-3 days interval in a dose of 2 billion microbe cells at the first, and 4 billion microbe cells at the second, feeding, being combined with drinking water.

This invention relates generally to the field of veterinary medicine,but more particularly to a novel live vaccine for the prevention ofsalmonellosis in water fowl, a process for making the same and itsprophylactic application in water fowl to combat salmonellosis caused bySalmonella typhi-murium.

S. typhi-murium caused salmonellosis affecting water fowl is marked byhigh incidence, very contagious character and often fatal outcome.

Furthermore, S. typhi-murium has been implicated as one of the principalagents causing food toxicoinfections in man while the water fowl is themajor host of the microbe in natural conditions and the productsobtained from slaughtering the infested poultry constitute the basicsource of infections in man.

Prevention and management of salmonellosis in water fowl reliesprimarily on the use of medicinal drugs and general veterinary-sanitarymeasures, which, as a rule, fall short of ensuring a higher healthstandard at the farm and maintenance of the livestock at a desirednumerical level. The specific prophylaxis of salmonellosis in water fowlis extremely inadequate.

There is known a polyvalent killed vaccine used for colibacillosis andsalmonellosis in fur animals, poultry calves, young pigs, which consistsof a four-billion suspension of S. typhi-murium, S. cholerae-suis, S.dublin and E. coli of 24 different serotypes killed with formalin andthimerosal. The vaccine, produced as a liquid, is recommended for thesubcutaneous administration repeated twice in birds: first in a dose of0.25-0.5, and then 0.5-1.0 ml. (Veterinary statute, Moscow, "Kolos"publishers, 1973, vol. I, p. 548; manual entitled "Veterinary agents,"Moscow, "Kolos" publishers, 1981, p. 223).

The vaccine exhibits a low immunogenicity. Inoculation does not conferthe complete protection against disease or prevent the individual frombecoming a carrier. Moreover, the vaccine contains Salmonella (S. dublinand S. choleraesuis) and Escherichia (0111, 0119, 0127, 0139, etc.),which are not pathogenic in birds. Twice repeated parenteraladministration of the vaccine with a syringe is too complicated for alarge scale inoculation campaign. The aforesaid vaccine poses asubstantial danger of allergic reaction.

Of the greatest promise in the prevention of salmonelloses in animalsare the live vaccines capable of stimulating the cellular mechanisms ofimmunity which play a leading role in the protection of the organismagainst infectious agents (Collins, Bact. Rev. 1974, 38, 4, 371-402).

Current practice makes use of the live vaccines for salmonellosis:calves of the strain S. dublin (U.S. Pat. No. 3,356,574 patented Dec. 5,1967), pigs of the strain S. cholerae-suis (U.S. Pat. No. 3,364,117 ofJan. 16, 1968), sheep of the strain S. abortus-ovis (French Pat. No.2,464,300 of Mar. 6, 1981). However, the live vaccines for salmonellosisin water fowl are not described in the literature.

An object of the invention is to provide a novel live vaccine which willbe highly immunogenic, efficacious, safe and simple to manufacture,convenient in use, having a long storage life, capable of establishing astrong immunological resistance to salmonellosis in water fowl.

The object is accomplished by providing a novel live vaccine for theprevention of salmonellosis in water fowl, consisting of the suspensionof a live culture prepared from the attenuated strain Salmonellatyphi-murium No. 3 deposited at the All-Union State Research ControlInstitute for Veterinary Preparations, the USSR Ministry of Agriculture(No. 121), in a concentration of 2-4 billion of microbe cells per 10 cm³of drinking water. To sustain the microbe cells in viable stage, thevaccine of this invention comprises a protective medium of the followingcomposition, in percent by weight:

saccharose: 4-12.0,

gelatin: 0.6-3.0,

water: the remainder

in an amount of 1 cm³ of the protective medium per 30-40 billion ofliving microbe cells.

What is new and useful likewise is a process for making the vaccine ofthis invention. The process for making the live vaccine for theprevention of salmonellosis in water fowl by means of growing the strainSalmonella typhi-murium in a culture medium containing sources ofcarbon, nitrogen, mineral salts and biologically active substances toproduce the highest attainable accumulation of biomass with subsequentdrying and obtainment of the vaccine, according to the invention,utilizes the strain Salmonella typhi-murium No. 3, deposited at theAll-Union State Research Control Institute for Veterinary Preparations,the USSR Ministry of Agriculture, as the growth culture, said process ofgrowth being effected at a temperature of 37°-38° C. for the duration of10-14 hrs. Prior to drying the biomass is preferably mixed with aprotective medium of the following composition, percent by weight:saccharose 4-12.0, gelatin 0.6-3.0, the balance being water, in anamount of 1 cm³ of the protective medium per 30-40 billion of microbecells.

The vaccine of this invention is innocuous, possesses high immunogenicpotential and in oral administration can be employed as a means ofspecific prophylaxis against salmonellosis in water fowl, e.g. atunfavourably affected farms.

The attenuated strain S. typhi-murium No. 3 used to produce the vaccineis distinguished by the fact that its genome comprises two mutationswhich, independently of each other, act to reduce virulence and ensure asteadily low residual virulence.

The attenuated strain is characterized by the following features:

morphological features: appears as small highly motile gram-negativerod-shaped microorganisms having no spores or capsules and measuring1.5-2 μm×0.3-0.5 μm;

culture properties: produces a uniform clouding in the meat-peptonebroth after 8-10hours of growth at a temperature of 37° C., i.e.exhibits a growth rate somewhat less than that of the virulentSalmonella strains which produce marked clouding of the broth in 4-5hours. On the meat-peptone agar forms transparent, circular, smooth,slightly convex colonies (S-shaped) of a fine granular structure 1-3 mmin diameter. The strain is resistant to streptomycin (300 units/ml);

Fermentation properties: ferments glucose, maltose, mannitol, arabinose,sorbite, xylose, producing acid and gas; forms H₂ S, does not fermentsaccharose, lactose, raffinose, does not curdle milk, does not dilutegelatin, does not form indole;

Antigen structure: agglutinates under the influence of the monoreceptor"O" serums: 1,4,5,12 and H-serums of the first phase (i) and secondphase (1, 2).

The attenuated strain selected from the population of the streptomycinsensitive virulent strain S. typhi-murium No. 415 closely resembles theparent strain in morphological aspects, fermentation properties, and theantigen structure. Of the culture properties uniquely pertaining to theattentuated strain as contrasted to the epizootic variety, are a lowreproduction rate and high resistance to streptomycin.

The residual virulence of the strain expressed in LD₅₀ for white miceweighing 14-16 g after intradermal inoculation is 8.08×10⁸ ±1.28×10⁸,LD₅₀ of the starting (virulent) strain being 18.9±8.4 microbe cells.Therefore, the residual virulence of the attenuated strain in white miceis 4.0×10⁷ times lower than that of the starting strain.

Stability of the residual virulence has been ascertained through aseries of ten passages in white mice and five passages in calves. Theresidual virulence of the attenuated strain did not change afterrepeated passages. The data are summarized in Table I.

                  TABLE I                                                         ______________________________________                                        LD.sub.50 of S. typhi-murium strains prior to and                             following passages                                                                             LD.sub.50 after                                                               LD.sub.50 series of 10                                                                            series of 5                                               prior to  passages in                                                                             passages in                              NOS. STRAINS     passage   white mice                                                                              calves                                   1    2           3         4         5                                        ______________________________________                                        2    Attenuated  8.08 × 10.sup.8                                                                   1.0 × 10.sup.9                                                                    7.94 × 10.sup.8                    3    Virulent    18.9      10        10                                       ______________________________________                                    

The difference of LD₅₀ value for the attenuated strain prior to andfollowing passages is statistically negligible (P<0.01).

The immunogenic capacity of the attenuated strain compared to a heatedvaccine prepared from the virulent strain was studied on white miceweighinhg 14-16 g subjected to a single immunization with subcutaneousdose of 1 million microbe cells. 21 days following the immunization themice were infected with the virulent strain in doses of 0.01, 0.1, 1 and10 thousand microbe cells assigning 10 animals per one dose. Theimmunogenic capacity was evaluated by the efficacy index essentiallybeing a ratio of the LD₅₀ in the study group to that in the control.

The immunogenic capacity of the attenuated strain was on the average 9times that elicited from killed cells of the virulent strain (efficacyindices 92 and 10.4, respectively).

What follows is a detailed description of the process for preparing thevaccine of this invention.

The culture is grown in reactors in a culture medium such as Hottinger'sbroth for 10-12 hours at a temperature of 37°-38° C., being constantlymixed (50-60 r.p.m.) and continuously aerated to produce enrichment ofeach liter of the culture medium with air in the proportion of 1:2.

The thus grown broth culture is precipitated by centrifuging, thendiluted in 18-19 l bottles with a protective medium to a concentrationof 100-120 billion microbe cells according to the visual opacitystandard, placed in ampoules and dried by sublimation in vacuum, andfinally sealed. The living bacteria count per 1 cm³ of the vaccinesubsequent to drying is 30-40 billion.

Determination of the living Salmonella count per 1 cm³ of the vaccine isperformed by the following method. Two samples, each comprising threeampoules of the vaccine, are prepared. The vaccine in ampoules isdiluted to the initial volume with physiological saline solution, thenmixed and evaluated for Salmonella concentration in both samples by theopacity standard. The bacterial concentration in the vaccine should be100 billion of microbe cells per 1 cm³.

Both samples are then used to prepare tenfold dilutions with separatepipettes to 10⁻⁹ in a sterile meat-peptone broth. From the 10⁻⁸ and 10⁻⁹dilution of both samples 0.1 cm³ provides by each are placed into fivedishes with meat-peptone agar. The culture material is uniformlydistributed by tilting the dish. The dishes are then placed in athermostat at a temperature of 37° C. for a period of 24 hours,whereupon the number of grown colonies and the concentration of livingSalmonellas (x) in billion per 1 cm³ are determined by the formula:##EQU1## where x is the concentration of living Salmonellas per 1 cm³,

p is the number of colonies in all the dishes containing the 10⁻⁸dilution,

q is the number of dishes with 10⁻⁸ dilution,

p_(I) is the number of colonies in all the dishes containing the 10⁻⁹dilution,

q_(I) is the number of dishes with 10⁻⁹ dilution.

Concentration indices for each sample are summed up and divided by thenumber of samples, thereby arriving at the mean concentration of livingSalmonellas in the vaccine, expressed in billions of microbe cells per 1cm³.

For safety determination, the desiccated vaccine extract is dissolved inphysiological salt solution to a concentration of 20 million microbecells per 1 cm³ according to the visual opacity standard andsubsequently administered to white mice weighing 16-18 g subcutaneouslyin the dorsal region utilizing a dose of 10 million microbe cells in avolume of 0.5 cm³. The vaccine is pronounced safe if of the original 10mice 8 are still alive at the end of a ten day period.

The test of the vaccine's activity requires the use of three ampoules.Each ampoule with the vaccine is filled up with physiological salinesolution to the starting volume. The completely dissolved vaccine isthen transferred into a sterile tube, the common sample being used toprepare a dilution of the vaccine in physiological saline solution at aconcentration of 300 million living bacteria per 1 cm³. 350-400 g areselected, of which number 10 individuals receive the vaccinesubcutaneously into the inguinal region in a dose of 300 million livingmicrobe cells per 1 cm³. After 14-16 days, both the immunized and 10control guinea pigs are infected with a lethal dose of a titratedculture of the control strain S. typhi-murium. Observation of theexperimental animals continues 10 days following the death of half ofthe controls. Over the period of observation all or at least 8 guineapigs from the control group should die while of the ten immunizedanimals at least 8 should survive.

In use the vaccine is a suspension of microbe cells of the attenuatedstrain S. typhi-murium No. 3 in a concentration of 2-4 billion livingbacteria per 10 cm³ of drinking water. The vaccine is employed in theprevention of Salmonellosis in water fowl. for example, the young ofducks and geese, at unfavourably affected farms. The vaccine is given toducklings and goslings starting from the third day of life, beingadministered orally at a two days interval in a dose of 2 billion at thefirst, and 4 billion microbe cells at the second, feeding. Immunity isinduced 3% days following administration of the second dose of thevaccine and is retained as long as six months.

Further illustrative of the invention are the following examples ofmaking and testing the vaccine.

EXAMPLE 1 Preparation of a single series of the vaccine

This process for making the vaccine comprises the following steps:

obtainment of a growing culture. To this end, an ampoule containingdesiccated attenuated strain S. typhi-murium No. 3 was emptied into twoor three 100-200 cm³ flasks with Hottinger's broth (culture volume onehalf of flask volume) accompanied by a control culture into the beakerscontaining meat-peptone agar, meat-peptone broth, meat-peptone liverbroth under petroleum jelly. Cultivation was conducted for 14-16 hoursat a temperature of 37° C. The culture was inspected for impurities bymicroscopic examination of the smears stained according to Gram'smethod. The pure culture was placed into 200 cm³ flasks containingHottinger's broth allowing 10 cm³ of the culture per 100 cm³ of themedium, and subjected to growth at a temperature of 37° C. for 14-16hours. The number of flasks under cultivation was dependent upon thedesired volume of the matrix culture. Control cultures were performed ina meat-peptone agar, meat-peptone broth, meat-peptone liver broth (MPA,MPB, MPLB) under petroleum jelly.

To obtain the matrix culture, the contents of one flask were transferredinto a bottle containing 10 l of Hottinger's broth with simultaneouscontrol culture in beakers with MPA, MPB, MPLB under petroleum jelly,and in Petri dishes containing Endo medium in order to avoidcontamination by extraneous microflora.

The cultivation in the bottles was carried out for 10-12 hours at atemperature of 37° C. in an amount required for cultivation in thereactor. The thus obtained growth was used for cultivation in beakerscontaining culture mediums or in Petri dishes containing Endo medium,and also for microscopy and determination of the concentration ofmicroorganisms according to the visual standard, said concentrationexpected to be at least 500 million microbe cells per 1 cm³.

The growing culture, screened for absence of impurities, was placed inthe reactor containing a sterile culture medium cooled to 38° C. toestablish a proportion of 8-10% of culture to the overall medium volumewhile simultaneously adding 0.1% (by weight of dry substance) of 40%sterile glucose solution.

Cultivation in the reactor continued for 10-12 hours at a temperature of37° C., the culture being constantly mixed (50 r.p.m.) and continuouslyaerated to produce enrichment of each liter of the culture medium in theproportion of 1:2 per minute.

While performing cultivation, a sample of culture was taken every 1-2hours to ascertain pH, purity of growth and concentration of microbecells. In the case of elevated pH during cultivation, the culture wasadded with 40% sterile glucose solution (0.1% by weight of drysubstance). Cultivation process was terminated on reaching the stablestage when the living microbe count ceased to increase.

The thus obtained culture was cooled to 6°-8° C. and tested forimpurities by microscopy and performing culture in beakers with MPA,MPB, MPLB under petroleum jelly or in dishes with Endo medium. Theculture contained 30-40 billion microbe cells per 1 cm³ according to thevisual standard, had a pH value between 7.4 and 7.6, and agglutinatedunder the influence of monoreceptor "O" serums: 1, 4, 5, 12 and H-serums(i, 1, 2). Subsequent to cooling the pure broth culture was transferredvia a sterile tubing for concentration in the supercentrifuge.

To maintain the sterility conditions, the rotor of the centrifuge wastransferred into the manual compartment, wherein the cultivatedbacterial mass was collected from the rotor into the previously weighedand sterilized three liter jars containing beads. Subsequently, each jarwas weighed again, the difference of weight giving the actual amount byweight of the collected concentrated bacterial culture.

The thus collected bacterial culture was diluted in 18-20 liter bottleswith a protective medium to a concentration of 100-120 billion microbecells per 1 cm³ according to the visual standard of opacity.

The protective medium was prepared by the following sequence ofprocesses: boiling distilled or demineralized water was used to dissolvegelatin in the strength of 1.5-2%, whereupon it was added with 10% ofsaccharose, subjected to boiling for a maximum of 5-6 minutes,subsequently strained through a cloth filter and given a pH value of7.6-7.8 by adding a 4% solution of caustic soda, and then sterilized ata temperature of 110° C. for 30 minutes. Subsequent to sterilization theprotective medium was tested for sterility by subjecting it to culturein MPA, MPB, MPLB under petroleum jelly.

The microbic culture suspension thoroughly mixed with the protectivemedium was placed in sterile ampoules and stoppered with loose cottontampons. While being dispensed the vaccine was continuously mixed. Thevaccine containing ampoules were inserted into a magazine to be putunder freeze-storage in the refrigerator at a temperature of -40°-50° C.for 8-14 hours.

Upon completion of freezing the vaccine containing ampoules weretransferred into the sublimation device, wherein they were subjected todrying.

Completion of the drying process was marked by the attainment of thegiven terminal temperature of the cultivated material (25° C.), minimalpressure inside the chamber, said values being maintained the same overthe final hours of drying. Activation of the shelf heating, vacuumpumps, and discharge of the dried vaccine were performed in compliancewith the operating instructions attached to the sublimation unit.

Ampoules with the dried vaccine were sealed on a drum collector unit ata maximum pressure of 0.2 mm Hg. The sealed ampoules were testedmacroscopically for the presence of foreign impurities, while the stateof vacuum inside the ampoules was determined using the instruments ofD'Arsonvalle's or Tesla's design. The ampoules were then designated withlables listing the name of substance and serial number.

The ampoule contained vaccine was subjected to the following tests:

appearance,

purity and culture pattern,

visual concentration per 1 cm³,

living microbe cell count and number of vaccine doses per 1 cm³,

moisture content in percent by weight,

solubility,

presence of vacuum in ampoules,

operational safety,

immunogenicity.

EXAMPLE 2

Laboratory test of the living vaccine. Safety of the vaccine was testedon 2-5 day old ducklings and goslings in subcutaneous administrationusing doses of 1,2,5,10 and 2,5,10,20 billion of microbe cells,respectively.

The survival time of the vaccine strain in the animal's body wasdetermined by bacteriological investigation of the internal structuresand tissues of the vaccinated birds conducted at an interval of 5-10days for a period of 2 months subsequent to the inoculation.

The immunogenic properties of the vaccine were studied in subcutaneous,combined (subcutaneous and oral), as well as oral administrationperformed once or twice. In subcutaneous administration the vaccine wasintroduced in a dose of 2 billion, whereas in oral and combined mole 1and 2 billion of microbe cells were given at two days interval.

The experiments showed that the live vaccine, whether givensubcutaneously or twice orally in the above-mentioned doses, did notproduce any untoward reaction in 2-5 days old ducklings and goslings,control slaughtering of the birds 5, 10, and 20 days followinginoculation showed no evidence of pathological changes in the internalstructures.

In oral technique of immunization, complete excretion of the microbecells occurred by the 35th day in ducks, and by the 40th day in geese.

The results of experimental studies of immunogenic properties of thelive vaccine in diverse modes of application as compared to the knownkilled vaccine were listed in Tables 2 and 3. As seen in Table 2, 15days subsequent to inoculation with the live vaccine administered in anyof the modes under study, the ducklings were rendered immune to asubcutaneous infection with the virulent strain S. typhi-murium in adose of 3 LD₅₀, the coefficient of immunologic efficacy (CIE) being 100.

At the same time, a single oral vaccination conferred immunity of asomewhat lesser intensity than the twice repeated administration. Thus,for example, 15 days following inoculation consisting of a singleadministration, the CIE value was 100 in response to 3 LD₅₀ infection,and 80 and 60 after a lapse of 30 and 45 days, respectively, in responseto 6 LD₅₀ infection with the virulent strain.

The killed vaccine exhibited considerably less potent immunogenicproperties.

The vaccine immunogenic capacity in the experiments on young geese(Table 3) was also sufficiently pronounced. CIE value 15, 30, 45, 60days following twice repeated oral vaccination was 80, 80, 73, 73,respectively, in response to 3 LD₅₀ infection with the virulent strain.

The corresponding indices for the killed vaccine were 48, 33, and 10.

EXAMPLE 3

A test of the live vaccine under the industrial conditions. Anindustrial trial series with the vaccine were carried out at the poultryfarms adversely affected by salmonellosis. The inoculation was performedaccording to the above-mentioned scheme on a total number of 107,400young ducks. The known killed vaccine, whose administration adhered tothe appropriate instructions, was used for comparison.

The mean results of a series of three industrial trials were summarizedin Table 4. Survival rate in the study groups inoculated with the liveor killed vaccine was on the average 96.5% and 88%, respectively,whereas this value in the control group was 83%. The indices of weightgain in poultry were 42.7, 36.0, 34.8 g, respectively, while the costsper each 100 kg of weight gain were 4.5, 5.8, 6.6 feed units.

Differences of the immunogenic efficacy indices exhibited by the liveand killed vaccines were statistically authenticated (P<0.01).

The general industrial saving accrued from the prevention of mortalityin poultry, increased weight gain, lower costs of feeding andmedication.

                                      TABLE 2                                     __________________________________________________________________________    Immunogenic properties of the vaccine in ducklings                            __________________________________________________________________________                 Infection subsequent to inoculation (day)                                     15           30           45                                             Immuni-                                                                            number       number       number                                         zation                                                                             of   infection                                                                             of   infection                                                                             of   infection                         Nos                                                                              Vaccine                                                                            technique                                                                          birds                                                                              dose CIE                                                                              birds                                                                              dose CIE                                                                              birds                                                                              dose CIE                          1  2    3    4    5    6  7    8    9  10   11   12                           __________________________________________________________________________    2  Vaccine                                                                            single                                                                             20   3LD.sub.50                                                                         100                                                                              30   10LD.sub.50                                                                        100                                                                              30   15LD.sub.50                                                                        100                             of this                                                                            subcuta-                                                                 invention                                                                          neous                                                                         oral-                                                                              20   3LD.sub.50                                                                         100                                                                              30   10LD.sub.50                                                                        100                                                                              35   15LD.sub.50                                                                        100                                  subcuta-                                                                      neous                                                                         single                                                                             20   3LD.sub.50                                                                         100                                                                              25    3LD.sub.50                                                                         80                                                                              30   6LD.sub.50                                                                          60                                  oral                                                                          double                                                                             20   3LD.sub.50                                                                         100                                                                              30   10LD.sub.50                                                                        100                                                                              35   15LD.sub.50                                                                        100                                  oral                                                                  3  Known                                                                              double                                                                             20   3LD.sub.50                                                                          52                                                                              20   10LD.sub.50                                                                         40                                                                              20   15LD.sub.50                                                                         20                             killed                                                                             subcuta-                                                                 vaccine                                                                            neous                                                                         double                                                                             20   3LD.sub.50                                                                          48                                                                              20   10LD.sub.50                                                                         33                                                                              20   15LD.sub.50                                                                         10                                  oral                                                                  4  Control   10   3LD.sub.50                                                                         -- 10    3LD.sub.50                                                                        -- 10   3LD.sub.50                                                                         --                           __________________________________________________________________________                              Infection subsequent to inoculation (day)                                     60           90                                                          Immuni-                                                                            number                                                                             infec-  number                                                      zation                                                                             of   tion    of   infection                                      Nos                                                                              Vaccine                                                                            technique                                                                          birds                                                                              dose CIE                                                                              birds                                                                              dose CIE                                       1  2    3    13   14   15 16   17   18                           __________________________________________________________________________                 2  Vaccine                                                                            single                                                                             20   3LD.sub.50                                                                         100                                                                              20   3LD.sub.50                                                                         100                                          of this                                                                            subcuta-                                                                 invention                                                                          neous                                                                         oral-                                                                              20   3LD.sub.50                                                                         100                                                                              20   3LD.sub.50                                                                         100                                               subcuta-                                                                      neous                                                                         single                                                                             --   --   -- --   --   --                                                oral                                                                          double                                                                             20   3LD.sub.50                                                                         100                                                                              20   3LD.sub.50                                                                         100                                               oral                                                                  3  Known                                                                              double                                                                             --   --   -- --   --   --                                           killed                                                                             subcuta-                                                                 vaccine                                                                            neous                                                                         double                                                                             --   --   -- --   --   --                                                oral                                                                  4  Control   10   3LD.sub.50                                                                         -- 10   3LD.sub.50                                                                         --                           __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Immunogenic properties of the vaccine in goslings                                            Infection subsequent to inoculation (day)                                     15          30          45          60                                        number                                                                             infec- number                                                                             infec- number                                                                             infec- number                                                                             infecti-                      Immunization                                                                         of   tion   of   tion   of   tion   of   on                    Nos                                                                              Vaccine                                                                            technique                                                                            birds                                                                              dose                                                                              CIE                                                                              birds                                                                              dose                                                                              CIE                                                                              birds                                                                              dose                                                                              CIE                                                                              birds                                                                              dose                                                                              CIE               1  2    3      4    5   6  7    8   9  10   11  12 13   14  15                __________________________________________________________________________    2  Vaccine                                                                            single 10   3LD.sub.50                                                                        80 15   3LD.sub.50                                                                        60 15   3LD.sub.50                                                                        60 15   3LD.sub.50                                                                        60                   of this                                                                            subcuta-                                                                 invention                                                                          neous                                                                         double 10   3LD.sub.50                                                                        80 15   3LD.sub.50                                                                        80 15   3LD.sub.50                                                                        73 15   3LD.sub.50                                                                        73                        oral                                                                  3  Known                                                                              double 10   3LD.sub.50                                                                        30 15   3LD.sub.50                                                                        22 15   3LD.sub.50                                                                        12 --   --  --                   killed                                                                             subcuta-                                                                 vaccine                                                                            neous                                                                         double 10   3LD.sub.50                                                                        25 15   3LD.sub.50                                                                        20 15   3LD.sub.50                                                                         7 --   --  --                        oral                                                                  4  Control     10   3LD.sub.50                                                                        -- 10   3LD.sub.50                                                                        -- 10   3LD.sub.50                                                                        -- --   --  --                __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Results of industrial trials of the vaccine on ducklings (average of a        series of three tests)                                                                       Number of               Feed consumption per                           Immunization                                                                         ducklings                                                                           Mortality                                                                            Survival                                                                           Mean daily                                                                          each 100 kg of weight                  Nos.                                                                             Vaccine                                                                            technique                                                                            under study                                                                         number                                                                             % %    weight gain                                                                         gain, feed units                       1  2    3      4     5    6 7    8     9                                      __________________________________________________________________________    2  Live double oral                                                                          107400                                                                              3759 3,5                                                                             96,5 42,7  4,5                                       vaccine                                                                       of this                                                                       invention                                                                  3  Known                                                                              double sub-                                                                           3000  360 12                                                                              88,0 36,0  5,8                                       killed                                                                             cutaneous                                                                vaccine                                                                    4  Control                                                                            --      34800                                                                              5916 17,0                                                                            83,0 34,8  6,6                                    __________________________________________________________________________

We claim:
 1. A live vaccine for the prevention of Salmonellosis whichcomprises a suspension of live attenuated strain Salmonella typhi-muriumNo. 3 and a biologically acceptable carrier.
 2. The vaccine compositionof claim 1 wherein the strain is from that deposited at the All-UnionState Research Control Institute for Veterinary Preparations Decoratedwith the Order of the Red Banner of Labor Under No.
 121. 3. The vaccinecomposition of claim 1 wherein the strain is capable of growing in aculture medium having a high content of Streptomycin.
 4. The vaccine ofclaim 1 comprising a concentration of microbe cells of from 1×10¹¹ to2×10⁷ per cubic centimeter.
 5. The vaccine of claim 4 comprising waterand a concentration of microbe cells of from 4×10⁸ to 2×10⁸ per cubiccentimeter.
 6. The vaccine composition of claim 1 wherein the strainferments glucose, maltose, mannitol, arabinose, sorbite, xyloseproducing acid and gas; forms H₂ S and does not ferment saccharose,lactose, raffinose.
 7. The vaccine composition of claim 5 wherein theantigen structure agglutinates under the influence of monoreceptor "O"serums: 1,4,5,12 and H-serums: of the first phase (i) and second phase 1and
 2. 8. The vaccine of claim 1 which has been freeze dried.
 9. Thevaccine of claim 1 wherein the genome of said strain comprises twomutations.
 10. The vaccine of claim 1 further comprising a protectivemedium to preserve viability of the microbe cells comprising thefollowing composition in % by weightsaccharose: 4-12.0, gelatin:0.6-3.0, water: remainingin an amount of 1 cubic centimeter per 30-40million living microbe cells.
 11. A process for making a live vaccinefor prevention of Salmonellosis in water fowl comprising cultivating astrain Salmonella typhi-murium No. 3 deposited at the All-Union StateResearch Control Institute of Veterinary Preparations, the USSR,Ministry of Agriculture as No. 121 in a culture medium containing asource of carbon, nitrogen, mineral salts and biologically activesubstances at a temperature of 37°-38° C. to produce the highestattainable accumulation of biomass, recovering said biomass and dryingsaid biomass to obtain said vaccine.
 12. The process of claim 11 whereinsaid biomass is mixed with a protective medium comprising, saccharose,gelatin and water in an amount of one cubic centimeter per 30-40 billionof live microbe cells.
 13. A method for immunization of water fowlagainst salmonellosis which comprises administering to the fowl orallyat a 2-3 day interval a first dose of 2×10⁹ microbe cells of liveattenuated strain Salmonella typhi-murium No. 3 in drinking water and asecond dose of 4×10⁹ microbe cells of live attenuated strain Salmonellatyphi-murium No. 3 in drinking water.